Redox biology is increasingly being recognized as a key signaling theme in the cellular physiology field. Research in redox biology is greatly hindered by the lack of methods for monitoring defined redox processes in cells and cell subdomains. This project addresses this technical issue by engineering genetically encoded redox biosensors that selectively report the localization and molecular dynamics of specific reactive oxygen species (ROS). The following four specific aims are pursued: 1. Develop genetically encoded superoxide (O2?-) sensors. 2. Engineer genetically encoded fluorescent probes for singlet oxygen (1O2). 3. Engineer genetically encoded fluorescent probes for hydroxyl radical (?OH). 4. Use the fluorescent toolkit to examine reactive oxygen/nitrogen species (ROS/RNS) in doxorubicin-treated tumor and healthy cells. The proposed work is expected to establish new capabilities for in vitro and in vivo detection of these highly reactive, underexplored ubiquitous molecules showing important roles in pathophysiology. We will also perform experiments to capitalize on the capabilities of our new fluorescent probes, in terms of multi-color time-lapse imaging and high selectivity toward particular ROS/RNS. We will use these new fluorescent biosensors to advance our understanding of the roles of ROS/RNS in the anticancer activity and the cardiotoxicity side effect of doxorubicin, a widely used chemotherapy drug. The study may shine light on new strategies to reduce the cardiotoxicity of doxorubicin without abolishing its anticancer ability. As UCR is one of only a few research universities in the U.S. that are also federally designated Hispanic-Serving Institutions, we will include undergraduate and graduate students from underrepresented groups in this project. The training they receive will broaden the participation of underrepresented groups in the STEM field.